Dr. Baker: Dr. Luft will you please come to the podium and thank you very much for coming, I understand your travel circumstances were adverse so thanks for your persistence.

Dr. Benjamin Luft: Thank you, I would like to thank the organizers for the opportunity to speak to this distinguished committee. My name is Benjamin Luft and I am a Professor of Medicine at the Stony Brook, I began working with Borrelia over twenty three years ago and I was led to over thirty calls to a hundred publications in this field alone. My research has spanned both the basic and the critical sciences and along with my colleagues I pioneered these set tracks of the treatment of Lyme disease. I also performed the earlier clinical laboratory experiments with Azithromycin, Azithromycin, Doxycycline, Amoxicillin and most recently with Tigecycline. Lyme disease has been vexing and problem for the past quarter of the century, not because it is a highly pathogenic disease associated with the significant mortality but rather the infection tends to be insidious very protean and at times debilitating. It is important to realize that microbial persistence is fundamental to the lifecycle of the organism. Borrelia has involved to develop strategies to persist the wide range of environments on to different that hostile circumstances from the tick to the mammal, often persisting for use at a time. After untreated human infection the organism is able to persist the patients only to recrudesce later in life.

The metabolic biochemical and genetic characteristic of the organism of the persisting organism has not been well delineated. Where the organisms persist in the mammalian host is not fully known. The issue of persistence is also central for our understanding of the adequacy or adequacy of our current therapeutics. When I read the current version of the guidelines I, I myself agreeing with much of it, however, my difference is became more apparent in the approach to the treatment of the failures this is the area that I actually became most interested in Lyme disease when the first studies were published by Alan Steere which showed that there was efficacy of therapy but there was just a very high value rate.

What do you do with the treatment failures and why do they fail? This problem has [indiscernible] [00:03:00] me since I first got involved with Lyme disease. In the discussion section of the guidelines and a quote on this early treatment less than ten percent individuals do not respond to antibiotic therapy as evidence by the presence of objective clinical manifestation and rarely is treatment, re-treatment required, in the discussion of the manifestations of Lyme disease especially neuroborreliosis and this is not healing with a chronic disease, symptoms persisted in thirteen to thirty percent of patients who receive therapy and it was often manifested by persistence if there was a [00:03:48] [indiscernible], pain, weakness, malaise and fatigue and the treatment recommendations emphasize that responsive treatment is usually slow and maybe incomplete, re-treatment is not recommended unless relapse is shown by objective measures.

When one reads the actual symptoms that are associated with failure of lack of treatment they are not objective, they are very subjective and I think that that’s one of the areas in

the guidelines where I have a bit of problems, is this emphasis on objective versus subjective, what you can induce in seeing by examining the patient rather than what you understand by listening to the patient and I think what I am going to do in demonstrating just two of my studies that I have done that I think that this is an inadequate way of approaching this disease.

The guidelines are never clear as there are cause in the persistence symptoms and that this [00:04:56] [indiscernible] patients instead great attention is given to the impossibility of the notion that symptomatic chronic deeper, [00:05:05] [indiscernible] exist despite recommended treatment. This will make Lyme disease unique among infectious diseases it would also help it as a business if this is a major area of my research is the persistence of this infection. I would now like to present two studies, a human trial and an animal trial.

The human trial was funded by Pfizer and was published in the Annals of Internal Medicine and the animal trial was funded by VIA and has not been published yet it was done with Steven Bartel well I think many of you know, Steve Bartel was a pioneer of the animal model of Lyme disease, he is a member of the Institute of Medicine, he is a distinguish professor at the University of California Davis. Both of these studies that I will present are negative studies that is the tested antibiotic did not work as we had hoped or expected but both gave us valuable insights into some of the characteristics of Lyme disease.

So the first study was a comparison of Azithromycin compared to Amoxicillin in the treatment of erythema migrans, this was again as I said a study that was funded by Pfizer, it was a multi centered, double blind randomized control trial and was published in 1996 in the Annals of Internal Medicine. Patients were randomized between Azithromycin and Amoxicillin, Azithromycin was give five hundred milligrams everyday or seven days, Amoxicillin was given five hundred milligrams every eight hours for twenty days, these were this, this regimen was selected by the pharmaceutical company and most likely had something to do with the marketing of Azithromycin. As you could see from this slide the baseline demographic and clinic characteristics of the patients that these patients were comparable, except that there was an increase in a number of multiple erythema migrans lesions in the Azithromycin group.

Well one more to the clinical manifestations of erythema migrans in these valuable patients and I think that this is a very important slide because I think what it does is it emphasizes what are the symptoms and signs of Lyme disease, how does Lyme disease manifest itself. In the Azithromycin group and the Amoxicillin group the signs and the symptoms of disease were roughly comparable but as you can see that only twenty percent of patients who had erythema migrans had signs of illness whereas the great majority of them had various symptoms associated with the [indiscernible] [0:08:30] and this included things, symptoms such as fatigue, joint pain, head ache, paresthesias all these very protean in there in how they manifest. Well in this study what we are able to show was that the Amoxicillin was superior to Azithromycin, when we looked at the complete response to therapy a seventy six percent of patients who received

Azithromycin has a complete response compared to eighty eight percent of patients who received Amoxicillin. When we then looked at the partial response twenty two percent of patients who received Azithromycin had a you know had a response, had a partial response compared to twelve percent of those who received Amoxicillin, if you have looked at the compared any response or either complete or partial it was statically no difference between the two groups, however, when you have compared complete response to two groups you found that Amoxicillin was significantly more effective than Azithromycin in the treatment of these patients. Now this I think is a very important finding because what it does it starts to distinguish between complete and partial response.

When one looks at the serologic results and the serology was done by Russell Johnson at the University of Minnesota and in these studies what – was that he did a basically an idolizing test and he studied these patients serially and what he, while when you compare those who had a positive serologic response compared to those who had a negative response, what one found was that patients who had a negative serologic response, those who did not now to the antibodies response to the organism thirty six percent of them had a partial response compared to only seventeen percent who had a positive serologic response and what you just begin to see is that there – is that immune system is working in concert with the antibiotics and being able to effect a complete response and this was significant statistically significant as well.

Well further more we look, began to look at patient responses by the treatment date at the day twenty as I sad before patients who had received Azithromycin were significantly more likely to have a partial response then those who received Amoxicillin. Thirty nine percent of patients who received Azithromycin had a partial response compared to only seven percent of patients who received Amoxicillin. Well what did this all have to do with whether the patient was ultimately going to relapse and in this study what we found was that patients who received Azithromycin were significantly more likely to relapse later on during the course of the follow up and this was for as long as six months of follow up.

However, when one looked at what the, at the serologic response of those who received Azithromycin only half of them, fifty percent of them were still positive at that time of relapse. So this is kind of I think that this percent perspective the whole issue I think that Dr. Baker was speaking of before as the difficulty in the diagnosis of this disease. So what can we say in conclusion, Azithromycin was significantly less effective than Amoxicillin I have been treating with [00:12:58] [indiscernible] preventing relapse. Patients who partially responded to therapy were more likely to relapse; patients that did not develop a serologic response Borrelia were more likely to have a partial response to treatment and were significantly more likely to relapse. Thirty to twenty four patients who fail therapy or relapse were sero negative at the time of relapse. The symptoms at the time of relapse or failure were protean and I think that that’s a very important point, I will go back to the slide, because what it says is that when patients actually relapse due to Lyme disease that they don’t develop the signs, the objective signs of the disease what they do is they continued to complain of symptomatology and this is what is most

problematic for those of us who are taking care of patients. This seems to be more problematic than the arthritis or the what we call the objective signs, what they are most often complaining of is the fatigue, the joint pains, the head ache, the muscle pains, the stiff necks, the fever, paresthesias etcetera and I think that this is important because I think there is an over emphasis in the guidelines in terms of objective disease, signs that you can elicit compared to subjective symptoms that the patient is actually complaining of and that when you put this in the appropriate context or the patients who you know as an acute disease that continues to comply over a period of time of this type of symptomatology, the physician has to take that seriously and the guidelines should reflect that.

The second study that would like to talk about is the study that I did with Steve Bartel and Steve Bartel has done many of these slides and this has to do with the persistence of Borrelia burgdorferi after our antibiotic treatment in an animal model. Now the reason why I bring this up is the second issue that I found problematic with the guidelines as that there was editorialization regarding biologic possibility. My opinion I don’t believe that that has any place in guidelines that has a place when in study section, that has a place at our meetings whether it’s [Zickak] or IBSA that has that – but I don’t think it necessarily has a place in the discussion of the treatment guidelines. Well the study that I began doing with Steve was that Steve had done, had written a series of rat anticipated and a series of groundbreaking work regarding the persistence of Borrelia burgdorferi following antibiotic treatment in mice, this was done both when he was at Yale as well as when he was at UC Davis and there are some problems or some questions as to some ethologic issues regarding the disease but I think that there is some very important seminal findings in regard to what he found.

Well my interest was when I began studies in a new drug Tigecycline against Borrelia burgdorferi the reason why I studied Tigecycline is that I am part of a large scale sequencing project, we are involved with sequencing about twenty gene ohms of Borrelia and my fellow was looking through the database and noticed that in the gene ohm Borrelia that there is are lot of efflux pumps which is not surprising for environmental type of organisms that they would have an efflux pump that would pump out various antibiotics and other toxins actually its commonly found in environmental organisms so I became very interested or you know as a fellow project I asked them to study the efficacy of Tigecycline against Borrelia Burgdorferi and we were able to show that indeed it have an efflux pump and that the efflux pump was active, it could be a few inhibitive, the activity of Tigecycline went up so I had because Steve had previously published this work on persistence I wanted to see whether Tigecycline would be a more efficacious drug in the treatment of Lyme disease and so what we did is we treated mice that were infected with the N40 strain of Borrelia with either Ceftriaxone or Tigecycline, the Ceftriaxone was given for twenty five days and the Tigecycline was only given for ten days, we had Dave Nicola at Harvard Hospital has done series of pharmical kinetic experiments for us with Tigecycline in the mouse so that we could find a good dose for treating the animal and we found that there were two doses one does that would be limit what we would see in humans and the other one would be four times that level so that we would give it a super normal dose of – in the treatment of this infection.

Now when we looked at what the, oops, go back this is the pharmical kinetic and we looked at the MIC of various drugs are used for the treatments of Borrelia when Amoxicillin the MIC is around point three nine microgram this is a N40 strain, Ceftriaxone point zero one two, Doxycycline point seven eight and Tigecycline point zero, zero six micrograms, however, what was more even more significant was the dramatic difference that we saw in the MBC of the organism, that Tigecycline was clearly the most, had the lowest MBC and not only did have the lowest MBC but it acted most rapidly, here in this slide you see the activity of these various drugs and their ability to kill the organism at twenty four hours, forty eight hours and seventy two hours, the T is Tigecycline and you can see that as early as twenty four hours of full incubation the organism is killed whereas with Ceftriaxone it reaches its maximal effect at seventy two hours. Doxycycline is bacteria static for this organism and therefore you can't kill it even with twenty five micrograms. So experimental design was as I mentioned Ceftriaxone was given at sixty milligrams per kilogram twice daily for five days and then subcutaneously for twenty five days, Tigecycline was given subcutaneous for ten days at a dose of twelve point five of fifty milligrams.

In Steve’s model he has three sets of animals that are tested, those that are pre immune one week of infection with Borrelia, those that are in the early immune phase which are, had been affected with Borrelia for three weeks and those with late persistence who were infected for four months.

Dr. Baker: Dr. Luft you have exceeded your time so could you sort of -.

Dr. Luft: Rather I will just wrap it because I will give you these slides and he showed was that basically and I will give you the summary slide and a persistent follow up of Borrelia was found in mice treated with two different bacteria cidal antibiotics, the persistent Borrelia was found to be viable and transmissible. The persistent form cannot be cultured, it was uncultured but could be identified by xenodiagnosis by techs that he had basically placed on the animal, by RTPCR of Borrelia transcripts and PCR Borrelia specific DNA, although the organisms persisted antibody levels decreased and these findings were very similar to what our human findings were. In the studies I just wanted to wrap up, it will take me just one more moment, there are several findings that are incontrovertible and we believe have a direct impact on how we think about the pathogenesis in treatment of Lyme disease.

First we demonstrated by treating mice with antibiotics, we convert the organism from being cultivatable, to being viable and non cultivatable, although non cultivatable it can be detected by RT by PCR and RTPCR and maintains its ability to be infectious skin mice by both ticket and tissue transplant. At this time we keep compelled to state what our findings did not establish, they did not establish whether these persistent forms exist in the post treatment syndrome in humans and if so whether they are the cause of these patients symptomatology, certainly it raises the specter that this occurs but that we, but we still have the tools that are available and which we are developing to be able to do this in a more depended manner. Another important issue is whether these persistent

organisms exert biologic effect in the host. What these are and whether this effect is and whether this effect is pathogenic, it would seem obvious that there must be some sort of pathogen host interaction in order to maintain the organism in its persistent state but these needs to be characterized. Lastly, therapeutic still needed to develop to affect a radical cure of animals infected with Borrelia.

Dr. Baker: Thank you very much for your testimony, unfortunately because its time there will be no time for questions for this presentation I will reiterate if we have time leftover in the afternoon because some people are a little less linked in their presentations, we can come back and ask you questions Dr. Luft.

Dr. Luft: If you want the, well you have a copy of the slides.

Dr. Baker: And we do have copies of your full presentation.